Efficacy of true cinnamon (Cinnamomum verum) leaf essential oil as a therapeutic alternative for Candida biofilm infections

Objective(s): The essential oil (EO) extracted from Cinnamomum verum leaves has been used as an antimicrobial agent for centuries. But its antifungal and antibiofilm efficacy is still not clearly studied. The objective of this research was to evaluate the in vitro antifungal and antibiofilm efficacy of C. verum leaf EO against C. albicans, C. tropicalis, and C. dubliniensis and the toxicity of EO using an in vitro model. Materials and Methods: The effect of EO vapor was evaluated using a microatmosphere technique. CLSI microdilution assay was employed in determining the Minimum Inhibitory (MIC) and Fungicidal Concentrations (MFC). Killing time was determined using a standard protocol. The effect of EO on established biofilms was quantified and visualized using XTT and Scanning Electron Microscopy (SEM), respectively. Post-exposure intracellular changes were visualized using Transmission Electron Microscopy (TEM). The toxicological assessment was carried out with the Human Keratinocyte cell line. The chemical composition of EO was evaluated using Gas Chromatography-Mass Spectrometry (GC-MS). Results: All test strains were susceptible to cinnamon oil vapor. EO exhibited MIC value 1.0 mg/ml and MFC value 2.0 mg/ml against test strains. The killing time of cinnamon oil was 6 hr. Minimum Biofilm Inhibitory Concentration (MBIC50) for established biofilms was <0.2 mg/ml for all test strains. SEM images exhibited cell wall damages, cellular shrinkages, and decreased hyphal formation of Candida. TEM indicated intracellular vacuolation, granulation, and cell wall damages. Cinnamon leaf oil caused no inhibition of HaCaT cells at any concentration tested. Eugenol was the abundant compound in cinnamon oil. Conclusion: C. verum EO is a potential alternative anti-Candida agent with minimal toxicity on the human host.


Introduction
The use of medicinal plants as a treatment option is an ancient practice and continues in the modern world. Ancient Asians including Sri Lankans, Indians, and Japanese people especially used these phytochemicals as their therapeutic agents. The use of medicinal plants as therapeutic alternatives has helped many populations that do not have any access to novel treatments and drugs of high cost and low availability. So, the use of plants for the treatment of diseases/infections has been empirically employed worldwide. In the past few years, with scientific advances, more research was conducted to introduce new compounds with medicinal properties, allowing scientists to find new, effective, alternative medicinal compounds with low side effects (1)(2)(3)(4).
Essential oils extracted from C. verum leaves and bark were used as antimicrobial agents for centuries. Though this plant has been shown to be of great economic and pharmaceutical-medicinal interest (5), proper evidencebased studies are not yet carried out to evaluate the efficacy of cinnamon oils as an antimicrobial therapeutic agent.
Biofilms are communities of microorganisms attached to a surface living in a matrix of extracellular material derived both from the cells themselves and from the environment. Those microorganisms are usually organized into a three-dimensional structure (6)(7)(8) in order to form this complex biofilm ecosystem. Many human microbial infections (more than 65%) are related to biofilm formation on implanted biomaterials and/or host surfaces (9,10). Biofilms are more resistant to most available antimicrobials compared with planktonic counterpart. Thus, it is important to introduce alternative biofilm controlling and eradication strategies in order to treat biofilm infections (11).
Although several in vitro microbiological studies with essential oil extracted from C. verum leaves show antimicrobial activities related to many bacterial species in the health care area, including those that exhibit multiple antimicrobial resistance, its antifungal and antibiofilm effects on Candida spp. are still not comprehensive.
The standard Candida stocks were maintained in 80% glycerol in an ultrafreezer at -80 °C . To reactivate stock organisms, they were subcultured in freshly prepared Saboraud Dextrose Agar (SDA, OXOID) culture medium and incubated aerobically at 37 °C for 24 hr.
The standard cell suspensions were prepared by adjusting the turbidity in accordance with the 0.5 McFarland scale, which was equivalent to the absorbance of 0.08-0.10 (600 nm) corresponding to 5×10 6 CFU/ml.
All experiments were done in triplicate on two different occasions.

Essential oil
The essential oil of C. verum leaves extracted using steam distillation was purchased from Romik Lanka Marketing Services, Moratuwa, Sri Lanka (WCC/3569). The C. verum essential oil was diluted to 32 mg/ ml in Tween 80 (0.05%) solution and Roswell Park Memorial Institute (RPMI) 1640 buffered with MOPS (3-(N-morpholino) propane sulfonic acid) followed by sonication, 1 cycle of 20 sec for antimicrobial assays and in vitro cytotoxicity. (17).

Effect of C. verum leaf essential oil in the vapor phase
Effects of C. verum leaf essential oil in vapor phase on planktonic Candida cells were qualitatively determined using the microatmosphere method described by Şerban et al. (2011) (18).
Standard suspensions of all test strains were prepared as explained previously and SDA plates were inoculated separately using a sterile cotton swab. A sterile filter paper disc moistened with the working solution (100 µl) of EO was attached to the lid of the Petri dish. Plates were sealed with a piece of parafilm and incubated overnight at 37 °C aerobically. Control plates were prepared without filter paper disks. The presence or absence of growth inhibition of inoculated Candida on agar surface was observed after 24 hr incubation.

Determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC)
According to Clinical and Laboratory Standards Institute (CLSI) M27-A3 (19), 100 µl/well of the two-fold dilutions of EO in RPMI 1640 with MOPS were prepared in 96-well polystyrene microtiter plates. Subsequently, 100 µl of the prepared standard fungal cell suspensions containing 1×10 6 cells/ml were added to the dilutions and incubated at 37 °C for 24 hr. The MIC point was determined by visual observation for the presence and absence of growth (turbidity of the suspensions).
MFC was defined as the lowest concentration of essential oil required to inhibit the fungal growth completely. For MFC determination, 5 μl of solutions from each well of the previous experiment was plated in freshly prepared SDA plates and incubated at 37 °C for 24 hr aerobically. The lowest concentration which did not show any Candida growth on the SDA surface after incubation was considered as MFC.
Negative control: test organisms without oil or reference antifungal agent. 120 mg/ml chlorhexidine digluconate: positive control.

Minimum biofilm inhibitory concentration (MBIC 50 )
A 96-well sterile flat-bottomed polystyrene microplate was seeded with 5×10 6 CFU/ml standard inoculum of each test organism (100 μl/well) followed by aerobic incubation for 24 hr at 37 °C . The plate was washed once with sterile Phosphate Buffered Saline (PBS), and 100 μl of the essential oil dilutions were added to the treatment wells separately. 100 μl of RPMI 1640 was added to the negative control wells instead of oil. The plate was then aerobically incubated for 24 hr at 37 °C . Biofilm viability was quantified using XTT assay (17,21).

Minimum biofilm eradication concentration (MBEC)
The CFU assay was performed on EO-treated biofilms to detect the MBEC of the oil on established 24 hr mature biofilms after the 24 hr treatment with different concentrations of C. verum leaf oil, as explained previously (17).

Post-exposure architecture of established Candida biofilms (scanning electron microscopy (SEM)
For determination of post-exposure architectural properties of Candida biofilms, mature biofilms were established on sterile 10 mm diameter glass coverslips and treated with 0.5, 1.0, and 2.0 mg/ml EO dilutions in RPMI 1640 for 24 hr as explained in a previous experiment and processed to examine under SEM (1,22).

Transmission electron microscopy (TEM) for determination of post-exposure cellular changes
To determine the effect of C. verum leaf oil on Candida cell structure, post-exposure cellular changes were visualized using a Transmission Electron Microscope (TEM) as follows.
Briefly, standard cell suspensions with 1×10 6 cell density were prepared in RPMI 1640. 1 ml of prepared suspensions were mixed with 9 ml of antifungal oil dilution in RPMI 1640 with the concentration of 10×MIC (10 mg/ml) separately (final EO concentration of the mixture was 1.0 mg/ml) and incubated at 37 °C for 24 hr aerobically. After incubation, the resulting cell suspension was centrifuged and the cell pellet was resuspended with the Karnovsky's fixative for 18-24 hours at 4 °C . After fixation, the specimen was processed as explained previously by Kapoor et al. (2017) (23).
Briefly, 96-well culture plates were seeded with cells (6.5×10 4 cells/ml and 100 µl/well) and incubated for 24 hr. Then the culture medium was replaced with fresh medium containing different concentrations of true cinnamon leaf EO. The treated plates were further incubated for 24 hr at 37 °C with 5% CO 2 . After the incubation, the viability of treated HaCat cells was quantified using an MTT assay (25).

Statistical analysis
Statistical analysis was carried out using the software, Statistical Package for Social Sciences (SPSS) version 16. Multiple means of more than three data sets were compared using one-way ANOVA and two-way ANOVA. The level of significance was taken at 5% (P<0.05).

Anti-Candida effect of C. verum leaf oil vapor
The results for antifungal efficacy of true cinnamon leaf oil vapor against three Candida test strains are presented in Table 1. Presence of growth inhibition on the agar surface indicates the sensitivity of relevant microorganisms to the treatment (18). All Candida test strains exhibited growth inhibition on the agar surface which is indicator of antifungal activity of C. verum leaf EO vapor on planktonic Candida (Table 1).

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC)
The MIC and MFC values corresponding to C. verum EO and the control antifungal chlorhexidine digluconate are represented in Table 2. C. verum exhibited similar efficacy as chlorhexidine digluconate on planktonic C. albicans (ATCC MYA-2876) and C. dubliniensis (ATCC MYA-646). C tropicalis (ATCC 750) was more susceptible for chlorhexidine digluconate compared with C. verum leaf oil. The emulsifier (Tween 80) did not affect the growth of the fungal strains at the used concentration. The MFC/MIC ratio showed that the C. verum leaf EO had a fungicidal effect on all three Candida species tested.

Killing time assay
This experiment determined the minimum time required for true cinnamon leaf oil (or positive control chlorhexidine digluconate) to eradicate the viable  Figure 1. According to obtained results, 0.5 mg/ml and 1.0 mg/ ml C. verum leaf oil do not kill Candida cells completely within 24 hr test period, whereas 2.0 mg/ml kill Candida cells completely within 6 hr.
2.0 mg/ml chlorhexidine digluconate has a rapid killing action on Candida cells compared to true cinnamon leaf oil hence killing time was 1 hr.

Minimum biofilm inhibitory concentration (MBIC 50 )
Minimum concentrations of the essential oil required to reduce the biofilm cell viability by 50% of negative control biofilms (biofilms without treatments) were defined as MBIC 50 . Figure 2 shows the percentage reduction of biofilm cell viability of 24 hr mature C. albicans (ATCC MYA-2876), C. tropicalis (ATCC 750), and C. dubliniensis (ATCC MYA-646) biofilms determined by XTT viability assay after 24 hr treatment with C. verum leaf oil compared with the negative control (biofilms without treatment).
According to obtained data from the XTT assay, concentrations required to reduce the biofilm viability by 50% are shown in Table 3.

Minimum biofilm eradication concentration (MBEC)
The minimum concentration of C.verum leaf oil required to kill the 24 hr mature biofilm completely (MBEC) was determined using CFU assay. Figure 3 represents the viability of 24 hr mature biofilms after treating with different concentrations of C. verum leaf oil and chlorhexidine digluconate for 24 hr determined by CFU assay. Table 4 shows MBEC values for mature C. albicans (ATCC MYA-2876), C. tropicalis (ATCC 750), and C. dubliniensis (ATCC MYA-646) biofilms.
TEM images of test strains after 24 hr exposure to MIC (1.0 mg/ml) of C. verum leaf oil and chlorhexidine digluconate were obtained as follows (Figure 7): TEM clearly indicates cell wall damages, intracellular granulations, and vacuole formation. Figure 8 shows percentage of growth inhibition after treatment with different concentrations. No significant change in HaCaT cell viability within the 0-1000 mg/ ml concentration range of true cinnamon leaf EO or chlorhexidine digluconate was detected. In this experiment, no toxic effect on HaCaT cells was noted with any concentrations of EO tested.

Discussion
Cinnamon has been used as a common culinary spice in several communities for centuries. Additionally, cinnamon has been also employed as folk medicine and phytomedicinal alternative in different cultures. The present study was conducted to find out the efficacy of C. verum/ true cinnamon leaf oil as a potential phytomedicinal therapeutic alternative against three commonest infection-causing Candida strains, namely C. albicans (ATCC MYA-2876), C. tropicalis (ATCC 750), and C. dubliniensis (ATCC MYA-646). The results of the present study show that C. verum EO had an important antifungal activity on all the tested strains of Candida.
In the present study, observations obtained from the microatmospheric plate method showed anti-Candida activity of C. verum leaf oil vapor. All three test strains exhibited a zone of growth inhibition with the administration of C. verum oil vapor (Table 1). Since, at normal body temperature, C. verum leaf oil vaporizes, it is important to evaluate the efficacy of an antimicrobial agent when it is not directly in contact with the affected areas. So this step can be considered as a screening test for anti-Candida activity of C. verum EO in its vapor phase.
The MFC/MIC ratio suggests that true cinnamon leaf EO had a fungicidal effect (26) on all three test strains. Though these data, in principle, demonstrate the antifungal action of C. verum leaf oil on planktonic Candida, MIC and MFC values of the current study are slightly different from recently published findings. (27,28). These differences in MIC and MFC may be due to the concentration variations of active compounds in EO used.
According to the classification of the activity level of plant materials based on MIC value introduced by Duarte et al. (2005), MIC up to 0.5 mg/ml is considered as strong active, concentrations from 0.55 to 1.5 mg/ ml are considered as moderately active, and above 1.5 mg/ml are considered as weakly active plant materials against tested microorganisms (29). According to this classification, the results obtained with the EO of C. verum leaves showed moderate antifungal activity on planktonic Candida. On the other hand commercial antifungal chlorhexidine digluconate exhibited a similar anti-Candida action as C. verum leaf oil for C. albicans and C. dubliniensis by demonstrating MIC value of 1 mg/ ml ( Table 2).  As well as minimum effective concentrations, the time required to completely eradicate/kill the microbial population is one of the key determinants of antimicrobial potency of a given antimicrobial agent and plays a major role in designing the dosage regime of a given antimicrobial drug (30). Results obtained from the killing time assay of C. verum leaf oil on test strain exhibited a concentration-dependent killing time. MFC (2.0 mg/ml) of cinnamon kills C. albicans within 6 hr whereas 2.0 mg/ml chlorhexidine digluconate kills C. albicans within 1 hr (Figure 1). Sub-MFC concentrations of C. verum EO does not exhibit any fungicidal effect within the 24 hr experiment period. Since killing time is concentration-dependent, shorter killing times can be acquired by increasing the concentration of EO, but the toxicological assessments should be contemplated. Though killing time is considered as an essential component of the antimicrobial profile, there is no evidence-based data on time-kill kinetics of C. verum leaf EO on Candida spp. The present study fills this deficiency of available data with an in vitro experimental design performed on ATCC type Candida strains.
In addition to the ability of true cinnamon leaf EO to act on Candida spp. planktonic cells, a strong antifungal potential was detected relative to the 24 hr established Candida biofilms. True cinnamon EO at a concentration of 0.2 mg/ml reduced the viability of C. tropicalis and C. dubliniensis biofilms by at least 50%, except for the C. albicans biofilm, which exhibited a 50% viability reduction with 0.1 mg/ml EO concentration (Table 3). These findings suggest the potential use of C. verum leaf EO as an antifungal for patients affected with chronic candidiasis. Notably, 24 hr exposure of 2 mg/ml EO caused complete killing of mature biofilms of the three types of investigated biofilms (Table 4). Though there are few studies in the literature on the antimicrobial effect of C. verum leaf EO, no published study assessed its action on Candida spp. biofilms. SEM images were taken to understand the structure of biofilms after subjecting to EO chemical stress. All biofilms of test strains exhibited cell wall damages, cell wall deformities, and leakages of intracellular materials with treatment of C. verum leaf oil (Figures 4, 5, and 6). Importantly, scanning electron microscopy confirmed the dose-dependent nature of the effects of cinnamon leaf EO. Fluconazole is a known antifungal agent which belongs to the azole group and inhibits synthesis of fungal sterol, ergosterol (20). On the other hand, chlorhexidine is a biguanide that is used as an antibacterial mouth rinse. It alters the morphology of cells, damages the cell wall of microorganisms, and releases intracellular components. It has been suggested as a well-known therapeutic antifungal agent for oral candidiasis (31,32). Since both antimicrobial agents have an effect on Candida cell wall, the ultrastructure of established Candida biofilms with MBEC of chlorhexidine digluconate and 0.008 mg/ml Fluconazole (maximum recommended in vitro assay concentration, CLSI) was also visualized. The intensity of the post-exposure response of Fluconazole was minimal due to low concentration.
To confirm the SEM observations, and to evaluate the intracellular/morphological changes of unicellular yeast forms, TEM images of C. albicans (ATCC MYA-2876), C. tropicalis (ATCC 750), and C. dubliniensis (ATCC MYA-646) test strains were taken after treatment with MIC of C. verum oil. TEM images showed cell wall damages and leakages of the intracellular compartment ( Figure 7). Importantly, TEM images revealed postexposure cytoplasmic changes including intracellular vacuoles formation, scattered cytoplasm, and formation of cytoplasmic granular inclusion bodies. These changes are suggestive of chemical stress on Candida cells by C. verum leaf oil.
The toxicity of true cinnamon EO on host tissues was evaluated using an in vitro cell culture model of the human non-cancer keratinocytes (HaCaT) cell line. Toxicology assessments provide proper understanding of effective, nontoxic dose and help in dose regimen designing by integrating both pharmacodynamics and pharmacokinetics of C. verum leaf oil. Furthermore, there is a lack of published data on toxicology studies of C. verum leaf oil and the current study fills that deficiency. The maximum concentration tested in this study was 1000 mg/ml (neat concentration of true cinnamon leaf EO oil) and none of the tested concentrations showed any inhibitory effect on HaCaT cells (Figure 8). This finding indicates the safe use of C. verum leaf EO up to 1000 mg/ml without any toxic effect on human cells.
Based on in vitro toxicology study results and pharmacodynamics of C. verum leaf oil on Candida sessile and planktonic cells, true cinnamon leaf EO can be considered a potential therapeutic alternative for Candida infections. Also, the authors suggest future research on the effect of C. verum leaf EO on a wide range of pathogenic fungi and bacteria.
The results of the chemical analysis of the true cinnamon leaf EO agree with the findings by previous authors, which indicate that eugenol is its most abundant chemical component (77.22%) (33,34) whereas the other chemical compounds appear in smaller concentrations.
According to available data, a similar mode of antifungal action of both cinnamon leaf oil and chlorhexidine digluconate was noted. Lipophilic eugenol can interfere with cell wall integrity and fluidity as well as biofilm extracellular matrix by actively penetrating the phospholipid bilayer of the cell membrane and ultimately disrupting it (35).

Conclusion
The EO extracted from C. verum leaves contains eugenol as its major chemical component. It possess an antifungal action on Candida spp. planktonic cells and biofilm by acting on the cell wall integrity. Moreover, it does not have any toxic effect on human keratinocytes.

Statement of Ethics
No human or animal experiments were performed in this research.

Author Contributions
GK-Conception or design of the work, data collection, data analysis, and interpretation, drafting the article, critical revision of the article, final approval of the version to be published; FC-data collection, final approval of the version to be published; TR and SNconception or design of the work, data collection, data analysis and interpretation, final approval of the version to be published; FJ, JP, GC, JD, and PL-data collection, final approval of the version to be published; JF-conception or design of the work, data collection, data analysis, and interpretation, drafting the article, critical revision of the article, final approval of the version to be published.